The case of bacteriophage PRD1, a membrane-containing dsDNA bacteriophage of the Tectiviridae family (33), will be used to illustrate the combination of cryo-EM and crystallographic data. Multiresolution imaging of the large (70-MDa, 700-A diameter) PRD1 particle resulted in a model for virion structure highlighting the structural elements involved in capsid stabilization and vertex dynamics during infection (13,17), as well as the structural parallels with the mammalian adenovirus (34). The atomic structure of PRD1 will soon be available (35,36), thus providing a direct comparison between the results obtained by multiresolution imaging and protein crystallography.

Figure 2 shows the main steps to be followed in obtaining a quasi-atomic model of a viral capsid. Essentially, the process starts with a manual fit of the

Fig. 2. Flow diagram showing the sequence of operations to be performed for obtaining a viral capsid quasi-atomic model and difference map.

atomic coordinates of the individual capsid proteins into the cryo-EM map of the complete capsid. When the virus under study is icosahedral, only those molecules included in the icosahedral asymmetric unit (AU) need to be manually fitted; the rest are generated later by symmetry operations. In a second step, the fitting is objectively optimized using the rigid body refinement approach. Each molecule in the AU is considered a rigid body, free to rotate and shift independently (as long as it does not clash with its neighbors) until the discrepancy between the atomic structures and the EM map is minimized. As the actual scale of the cryo-EM map is not known accurately, this is determined with the fitted crystallographic structure as a reference, and a second rigid body refinement is performed. At this point a complete capsid model can be created by applying icosahedral symmetry to the contents of the AU and used to analyze protein-protein contacts or to calculate a difference map.

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